BACHELORARBEIT

Effect of desalting on enzymes used for

energy reduction in pulp refining

VerfasserIn

Michael Josef Huber

Angestrebter akademischer Grad:

Bachelor of Science

Bachelorstudium: Lebensmittel- und Biotechnologie

Supervisor Georg Gübitz, Univ.Prof. Dipl.-Ing. Dr.techn

Co-Supervisor: Martin Nagl, Dipl.-Ing

Wien, 09. 09. 2022

Kurzfassung

Zellstoffveredelung ist sehr energieintensiv. Um diesen Energiebedarf zu reduzieren wird der Zellstoff
mit Veredelungsenzymen vorbehandelt. Vier solcher Enzymformulierungen wurden von der Industrie
zur Verfügung gestellt (EnzA, EnzB, EnzC und FiberCare R). In dieser Arbeit wurde untersucht wie sich
das Entsalzen auf die Aktivität dieser Enzyme auswirkt. Darüber hinaus wurde die Endoglucanase
EndoB des Enzymcocktails EnzB isoliert und untersucht. Zusätzlich wurde die Auswirkung der Enzyme
auf verschiedene Substrate untersucht. Zur Messung der Enzymaktivitäten wurden folgende
Methoden verwendet: CellG5 Endoglucanase Assay, Filterpapier Cellulase Assay, β-glucosidase Assay
und Xylanase Assay. Die Auswirkungen auf den Zellstoff wurden gemessen mit: HPLC, SEM, SEC und
FT-IR. Die spezifische Aktivität (U/mg) hat bei fast allen Enzymformulierungen durch das Entsalzen
zugenommen. Eine Ausnahme war FiberCare R wo die spezifische Endoglucanase Aktivität durch
Entsalzen abgenommen hat. Zwar hat die spezifische Aktivität im Allgemeinen zugenommen, die
volumetrische Aktivität (U/mL) ist jedoch gesunken, weil während des Entsalzens Proteine verloren
gingen. EnzC war die einzige Formulierung dessen volumetrische Aktivität durch das Entsalzen
zunahm. Die Untersuchungen am Zellstoff ergaben, dass alle Enzymformulierungen ähnliche
morphologische Veränderungen am Zellstoff herbeiführen, nämlich dünnere Fasern. EnzA, EnzB und
EnzC setzen hauptsächlich Xylose und Glucose frei, während FiberCare R in erster Linie Cellobiose
freisetzte. Bei der Untersuchung mit SEC konnte festgestellt werden, dass Behandlung mit EnzA, EnzB
und EnzC zu etwas kleineren Bruchstücken führte, während die Behandlung mit reinen
Endoglucanasen zu etwas größeren Bruchstücken führte. Zusammenfassend führt das Entsalzen zu
höherer spezifischer Aktivität. Diese Erkenntnis könnte die Basis für zukünftige industrielle
Anwendungen sein.

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Abstract

Pulp refining is very energy intensive. To reduce this energy requirement, the pulp is pretreated with
refining enzymes. Four such enzyme formulations were provided by the industry (EnzA, EnzB, EnzC
and FiberCare R). In this work, we investigated how desalting affects the activity of these enzymes.
Furthermore, the endoglucanase EndoB of the enzyme formulation EnzB was isolated and studied. In
addition, the effect of the enzymes on different substrates was investigated. The following methods
were used to measure enzyme activities: CellG5 endoglucanase assay, filter paper cellulase assay, β-
glucosidase assay, and xylanase assay. The effect on pulp was measured using HPLC, SEM, SEC and
FT-IR. The specific activity (U/mg) increased for almost all enzyme formulations due to desalting. An
exception was FiberCare R where the specific endoglucanase activity decreased by desalting.
Although the specific activity generally increased, the volumetric activity (U/mL) decreased because
of proteins loss during desalting. EnzC was the only formulation whose volumetric activity increased
due to desalting. The pulp studies showed that all enzyme formulations induced similar
morphological changes on the pulp, namely thinner fibers. EnzA, EnzB and EnzC released mainly
xylose and glucose, while FiberCare R released primarily cellobiose. When tested with SEC, it was
found that treatment with EnzA, EnzB and EnzC leads to slightly smaller fragments, while treatment
with pure endoglucanases leads to slightly larger fragments. In summary, desalting leads to higher
specific activity. This finding could be the basis for future industrial applications.

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0 Table of Contents
1 Introduction .......................................................................................................................................... 6
1.1 The pulp and papermaking process ............................................................................................. 6
1.2 Softwood and Hardwood pulp ..................................................................................................... 6
1.3 Pulping Processes ......................................................................................................................... 6
1.4 Refining enzymes .......................................................................................................................... 7
2 Materials and Methods ........................................................................................................................ 8
2.1 Materials........................................................................................................................................ 8
2.1.1 Enzymes and pulps ................................................................................................................ 8
2.1.2 Desalting ................................................................................................................................ 8
2.1.3 SDS-Page ................................................................................................................................ 8
2.1.4 β-Glucosidase assay ............................................................................................................... 9
2.1.5 Filter paper/Pulp Cellulase activity assay ............................................................................. 9
2.1.6 Chemicals for CellG5 Cellulase Activity Assay .................................................................... 10
2.1.7 XylX6 Xylose activity assay .................................................................................................. 10
2.1.8 Hydrophobic Interaction Chromatography (HIC) ............................................................... 10
2.1.10 Anion Exchange Chromatography (AEC) ........................................................................... 11
2.1.9 High Performance Liquid Chromatography (HPLC) ............................................................ 11
2.1.11 Chemicals for Size Exclusion Chromatography (SEC-MALLS) ........................................... 11
2.1.12 Chemicals for Bradford Assay ........................................................................................... 11
2.1.13 Laboratory facilities ........................................................................................................... 11
2.2 Methods ...................................................................................................................................... 12
2.2.1 Desalting .............................................................................................................................. 12
2.2.2 Enzyme Purification ............................................................................................................. 12
2.2.3 Protein concentration determination ................................................................................. 14
2.2.4 Activity Assays ..................................................................................................................... 14
2.2.5 Fourier-transform infrared spectroscopy (FT-IR) ............................................................... 15
2.2.6 Scanning Electron Microscopy (SEM) .................................................................................. 15
2.2.7 Size Exclusion Chromatography (SEC) ................................................................................. 15
2.2.8 High Performance Liquid Chromatography (HPLC) ............................................................ 16
3 Results and discussion ........................................................................................................................ 17
3.1 Basic characterization of the enzyme formulations .................................................................. 17
3.2 Basic characterization of purified EndoB endoglucanase ......................................................... 21
3.3 Enzymatic treatment of different pulps using EndoB ............................................................... 21
3.4 Enzymatic treatment of substrates with commercial enzyme formulations ........................... 22
3.4.1 HPLC ..................................................................................................................................... 22

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 3.4.2 Scanning Electron Microscopy ............................................................................................ 23
3.4.3 Size Exclusion Chromatography .......................................................................................... 25
3.4.4 FT-IR ..................................................................................................................................... 26
3.5 Composition of enzyme formulations........................................................................................ 30
4 Conclusion .......................................................................................................................................... 32
5 Appendix............................................................................................................................................. 33
Acknowledgements ............................................................................................................................... 35
6 References .......................................................................................................................................... 36
List of Figures......................................................................................................................................... 36
List of Tables .......................................................................................................................................... 37

5
1 Introduction
Refining is an essential step during pulp and papermaking. It increases the strength of the unrefined
pulp by inducing fibrillations, which in turn increase the fiber’s surface area. This strengthens the
fiber-to-fiber bond but makes the individual fiber weaker. Consequently, refining is a balancing act to
obtain the greatest possible paper strength. (Bajpai, 2018) Refining also ensures better water
absorption.
Refining is done primarily through the input of shear forces. This is very energy-intensive and
consumes 26.61% of the total electricity input during papermaking. (Haske-Cornelius et al., 2020)
To reduce energy consumption enzymes are added to the refining step. Several such commercially
available enzyme solutions, their activity and their effect on various pulps have been measured and
analyzed in this thesis.
For further clarity some background information about the papermaking process is given.
A detailed overview is given in figure 1.

1.1 The pulp and papermaking process

Raw materials undergo several steps on their way from plantation to paper. First, the wood is
debarked and chipped. Depending on whether hardwood or softwood is used, the paper will have
different qualities. Secondly, the pulping process is applied, which removes the lignin from the wood
chips turning the wood into a fibrous mass. Pulping is mostly done by two processes. The sulfate
(kraft) process and the sulfite process. They are described in more detail below. The pulp is then
refined to increase the strength and smoothness of the final product. The refined pulp is then
“dissolved” in water. The watery pulp gets pressed onto a form and the water is removed, resulting
in the final paper product.

Figure 1: Overview of Pulp and Paper manufacturing (Ochre Media Private Limited, 2018).

1.2 Softwood and Hardwood pulp

There are pulps made from softwood and pulps made from hardwood. Softwood pulps have long
fibers (LF) Hardwood pulps have short fibers (SF). Hardwood is denser and has a lower lignin content.
Softwood paper is more flexible, has high folding strength, high tensile strength, and has good
printability. Hardwood paper is looser, more opaque, and stiffer (Song, 2018).

1.3 Pulping Processes

During Pulping the wood chips are broken down, the fibers are liberated and dispersed in water and
turned into a web (i.e. paper). (Biermann, 1996) The so treated wood chips are called pulp. Pulping
can be done in a variety of chemical and mechanical ways. Currently two chemical pulping methods
are predominantly used for commercial pulping. Those are the Kraft process and the Sulfite process.

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Kraft pulping
During Kraft pulping the raw material is being cooked using sodium hydroxide and sodium sulfide at a
pH above 12 and a temperature of 160-180 °C under a pressure of around 8 bar for 0.5 to three
hours. This dissolves much of the lignin in the wood fibers. The advantages of the Kraft process are
that it is useful for any wood species, that it produces a high strength pulp (hence the name) and has
an efficient chemical and energy recovery cycle. Its disadvantages are that Kraft pulps are more
difficult to bleach, lower yields due to carbohydrate losses and that sulfur in its reduced form is
extremely odiferous. Kraft pulping is the most important pulping method by volume.

Sulfite pulping
In the sulfite pulping process mixtures of sulfurous acid or its salts are used to remove lignin. During
Sulfite pulping the wood chips are being treated with those chemicals at 120-150 °C under 5 to 7 bars
of pressure for 12-14 hours. The advantages of sulfite pulp are, that it is easily bleached, can be
refined relatively easily, can hold more water than kraft pulps and has a higher yield than Kraft pulps.
However, sulfite pulps are weaker than kraft pulp, the cooking cycle is longer, the chemical recovery
process is complicated and not all species of wood can be pulped easily. Sulfite pulping is the second
most important pulping method by volume. (Biermann, 1996)

1.4 Refining enzymes

As mentioned above enzymes are used during refining to decrease energy demand. Those enzymes
are cellulases and xylanases. Both act on components of pulp fibers. Cellulases degrade cellulose.
xylanases attack hemicellulose (xylan). They are either used together in complex enzyme
formulations or individually during refining.

Cellulase
Cellulose is the main component of wood and pulp fibers and consists of linear chains of β-1,4 linked
D-glucose units. Cellulases are enzymes; or rather multi-component enzyme systems, that cleave
cellulose. They consist of endo-β-1,4-glucanase, exo-β-1,4-glucanase and β-glucosidase.
Endoglucanases hydrolyze the β-1,4 glycosidic linkages on the amorphous part of cellulose. They cut
randomly within the chain. Exoglucanases attack on the reducing and non-reducing ends of cellulose
chains to release cellobiose. β-glucosidase hydrolyses cellulose chains released from endoglucanases
ans cellobiohydrolases, including cellobiose, from the non-reducing end, releasing glucose. (KEGG
ENZYME: 3.2.1.21, n.d.) Together they remove the primary wall and S1 layer of cellulose fibers.
Cellulase also fibrillates the S2 layer. This improves inter-fiber bonding and the tensile strength of
pulp. But cellulase treatment also results in loss of fiber intrinsic strength and negatively affects tear
strength.

Xylanase
Xylanase dissolves the xylan in hemicellulose, another component of wood. The removal of these
polysaccharides lets water molecules penetrate the cellulosic fiber. This may break some hydrogen
bonds connecting cellulose chains. Thus, making the fibers more flexible. Xylanases improve
accessibility for endoglucanases through degradation of xylan. (Kumar et al., 2021)

Aim of the study

The aim of the study was the investigation of the effect of salts on enzyme behavior. Commercial
enzyme formulations are available as complex formulations containing a high number of different
salts originating from the production process or added deliberately to stabilize the enzymes, which
can have positive or negative effects.

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2 Materials and Methods
2.1 Materials
2.1.1 Enzymes and pulps
Three different commercially available refining enzyme cocktails (EnzC, EnzB, EnzA) were provided by
companies of the Austrian pulp and paper industry. Furthermore, the commercial endoglucanase
formulation FiberCare R as well as the hydrolytic enzyme cocktail CTec3 were supplied by
Novozymes. The endoglucanase from the EnzB formulation was further purified. This purified
formulation shall henceforth be referred to as EndoB. The experiments were conducted with both
the unpurified enzyme formulations and EndoB. Additionally, experiments were repeated with
desalted versions of the commercially available enzyme mixes. The activities of the desalted and
salted enzyme mixes were later compared. Pulp samples were provided by paper manufacturers.
Those samples were the hardwood Sulfate (Short Fiber) pulps as well as long fiber softwood pulps LF
Sulfite and LF Sulfate. All used chemicals were of analytical grade and used without further
purification unless stated otherwise.

2.1.2 Desalting
Material and Buffers:

• 50 mM Citrate buffer pH 4.8 (calibration/elution buffer)

• PD-10 desalting columns
• 20% Ethanol (storage solution)

2.1.3 SDS-Page
Material and Buffers:

• 10x Tris/Glycine/SDS #1610732 (Running buffer)

• Protein Marker IV (pre-stained), peqGOLD #27-2110
• Laemmli Buffer (+33 µL Mercaptoethanol)
• Mini-Protean TGX Stain-Free Precast Gels 4-15%
• Mini-Protean Tetra Cell Biorad #1658005EDU
• PowerPac HV Power Supply Biorad #1645056

Solutions:

Staining Solution

• 600 mL H2O
• 300 mL EtOH
• 100 mL Acetic Acid
• 0.125% (1.25g/L) Coomassie Blue G-250

Destaining Solution

• 600 mL H2O
• 300 mL EtOH
• 100 mL Acetic Acid

Chemicals:

• Tris base
• Tris-Glycine-SDS Buffer 10 x
• Glycerol
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 • β-Mercaptoethanol
• Coomassie Briliant Blue R250
• Bromophenol blue
• Methanol
• Acetic acid
• Acrylamide/Bis-acrylamide
• Ammonium persulfate
• Tetramethylenedianmine
• Protein Marker peqGold Protein Marker IV (10-70 kDa, 10 bands)

2.1.4 β-Glucosidase assay

Materials and buffers

• 50 mM Citrate buffer pH 4,8 (enzyme reaction buffer)

• 500 mM Na-PO4 buffer pH 7 (color reaction buffer)

Solutions:

Substrate solution

• 2 mM 4-Nitrophenyl β-D-glucopyranoside in enzyme reaction buffer

• 2 mM 4-Nitrophenol stock (for calibration)

2.1.5 Filter paper/Pulp Cellulase activity assay

Materials and Buffers:

• 50 mM Citrate buffer
• Plate reader Tecan Infinite M200 PRO
• 96 well plate

Substrate

• 1% carboxymethylcellulose solution with a degree of substitution of 0.7 and an average

molecular weight of 90.000 g/mol (for calibration)
• 7.5 cm by 0.75 cm Whatman No. 1 filter paper (for Filter paper cellulase assay)
• 10 mg of various pulps (for Pulp cellulase assay)

Solutions:

DNS solution

• 390 mL MQ-water
• 2.5 g Dinitrosalicylic acid
• 4.0 g NaOH
• 75 g Rochelle salt (potassium sodium tartrate)

Stopping solution

• 1 M NaOH

Chemicals:

• NaOH (CAS: 1310-73-2)

• Dinitrosalicylic acid (CAS: 609-99-4)

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 • Rochelle salt (CAS: 6381-59-5)
• Glucose (CAS: 50-99-7, MW 180,16 g/mol)
• 1% carboxymethylcellulose solution with a degree of substitution of 0.7 and an average
molecular weight of 90.000 g/mol (CAS: 9004-32-4)
• TRIS (CAS: 77-86-1)
• Citrate (CAS: 77-92-9 (free of water), 5949-29-1 (Monohydrate))

2.1.6 Chemicals for CellG5 Cellulase Activity Assay

Materials and Buffers

• 50 mM Citrate buffer pH 4,8 (enzyme reaction/dilution buffer)

Solutions

Substrate solution

• 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside in 10% DMSO/H2O plus

sodiumazide (0.02% w/v) (3mL) mixed with Thermostable β-glucosidase (0.10 mL, 600 U/mL)
in 50% w/v ammonium sulphate solution plus sodium azide (0.02% w/v)

Stopping solution

• 2% (w/v) Tris buffer (pH 10)

2.1.7 XylX6 Xylose activity assay

Materials and Buffers:

• 50 mM Citrate buffer pH 4.8 (dilution buffer)

Solutions

Substrate Solution

• XylX6 colourimetric substrate and β-xylosidase plus sodium azide (0.09% w/w) dissolved in 5
mL mQ-water

Stopping Solution

• 2% Tris solution pH 10

2.1.8 Hydrophobic Interaction Chromatography (HIC)

Materials and Buffers

• 10 mM Acetate buffer pH 4.8, 1.5 M Ammonium Sulfate (Mobile Phase A)

• 10 mM Acetate buffer (Mobile Phase B)
• 20% Ethanol (storage solution)
• 5 mL HiTrap Phenyl HP columns

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2.1.10 Anion Exchange Chromatography (AEC)
Materials Buffers

• 10 mM Tris buffer pH 7.5 (Buffer A)

• 10 mM Tris buffer pH 7.5, 1 M NaCl (Buffer B)
• HiTrap DEAE 5 mL FF column
• 0.2 µm nylon filter tips
• Ethanol 20% (storage solution)

2.1.9 High Performance Liquid Chromatography (HPLC)

Materials and Buffers

• ION-300 column
• 0.45 µm nylon filter tips
• C1 solution (2% K4[Fe(CN)6] * 3 H2O)
• C2 solution (2% ZnSO4 * 7 H2O)
• mQ-water (for dilution)

2.1.11 Chemicals for Size Exclusion Chromatography (SEC-MALLS)

Materials and Buffers

• 50 mM NaNO3 buffer, 3 mM NaN3 (mobile Phase)

• 0.5 % carboxymethylcellulose CMC (Degree of Substitution: 0.9, Average molecular
weight= 250000 g/mol) in 50 mM citrate buffer pH 4.8 (substrate solution)
• 70% Ethanol (storage solution)
• 0.1 µm membrane filter
• 0.45 µm nylon filter tips

2.1.12 Chemicals for Bradford Assay

Materials and Buffers

• 50 mM Citrate buffer pH 4.8 (dilution buffer)

• 2 mg/mL BSA (calibration stock)
• Bradford solution supplied by Roth (RotiQuant)

2.1.13 Laboratory facilities

• Agilent 1260 Infinity II liquid chromatography system Agilent Technologies (USA)
• ÄKTA pure 25 protein purification system Cytiva (USA)
• Balance MSA 524S Sartorius Lab Instruments
GmbH (Germany)
• Balance MSE 1203S Sartorius Lab Instruments
GmbH (Germany)
• ChemieDoc Biorad (USA)
• Centrifugal Concentrator Vivaspin 20 GE Healthcare
MWCO 3000 Dalton (UK)
• Centrifuge 5427 R Eppendorf (Germany)
• Centrifuge 5920 R Eppendorf (Germany)
• Chromatography vials N11 Art.No. 702 01 HP Macherey-Nagel GmbH &
Co. KG (Germany)
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 • SI Analytics Refillable BlueLine 31 RX Redox (ORP) Electrode
Xylem (USA)
• HiTrap FF DEAE column Cytiva (USA)
• HiTrap Phenyl HP Columns Cytiva (USA)
• ION-300 column Macherey-Nagel (Germany)
• Leica EM ACE200 sputter coater Leica (Germany)
• Magnetic stirrer IKA C-MAG HS4 IKA (Germany)
• MALS HELIOS DAWN 2 detector Wyatt Technology (USA)
• Nanodrop photometer Implen NP80 Thermo Fisher scientific
(USA)
• Nylon filter tips 0.45 µm Sigma-Aldrich (USA)
• PD-10 Columns GE Healthcare (UK)
• FT-IR spectrometer Perkin Elmer Spectrum 100 PerkinElmer (USA)
• pH meter SevenCompact Mettler-Toledo (USA)
• Photometer Hitachi U2900 Hitachi (Japan)
• Pipettes Gilson (USA)
• PL aquagel-OH MIXED Guard (PL1149-1840, 8 μm, 7.5 × 50 mm2) pre-column Agilent
Technologies (USA)
• PL aquagel-OH MIXED H (PL1549-5800, 4.6 × 250 mm2, 8 μm) column Agilent
Technologies (USA)
• Power Pac HV Power Supply Biorad (USA)
• Pyrex glass tubes Corning (USA)
• Plate reader Tecan Infinite M200 PRO Tecan (Switzerland)
• Scanning electron microscope Hitachi TM3030 Hitachi (Japan)
• Thermomixer Eppendorf (Germany)
• Vortex VWR International (USA)
• Water bath GFL Type 1013 GFL (Germany)
• 96 Well plates Thermo fisher scientific
(USA)

2.2 Methods
2.2.1 Desalting
To compare the effect of salts on the activity of the commercially available enzyme cocktails, the
commercially available enzyme cocktails were desalted using PD-10 Desalting columns from GE
Healthcare. Each column contains 8.3 mL of SephadexTM G-25 Medium. First, the PD-10 columns
were filled with 25 mL equilibration buffer. The flow through, consisting of 20% Ethanol storage
solution, was discarded. Once the equilibration buffer had entered the column completely 2.5 mL
enzyme cocktail were added to the column. After the enzyme samples had entered the column as
well 3.5 mL elution buffer (50 mM citrate buffer pH 4.8) was added to the column. A test tube was
placed under the column and the flow through, consisting of the desalted enzyme cocktail, was
collected.

2.2.2 Enzyme Purification

A 10x concentrated EnzB formulation was purified using an ÄKTATM pure 25. During purification the
EnzB enzyme cocktail underwent purification with Hydrophobic Interaction Chromatography, was
concentrated and underwent another round of purification with Anion Exchange Chromatography.
Between the purification steps, SDS-PAGE of the collected fractions was performed to identify

12
desired enzyme fractions. Fractions with the desired protein content were pooled and further
purified. The individual purification steps are described with more detail below.

2.2.2.1 Hydrophobic Interaction Chromatography (HIC)

Three connected 5 mL HiTrap Phenyl HP columns were used for HIC purification. Mobile phase A
consisted of 10 mM Acetate buffer, pH 4.8 and 1.5 M Ammonium Sulfate. Mobile Phase B consisted
of 10 mM Acetate buffer. After the washing procedures were done 70 mL sample (3.5 mL 10x
concentrated EnzB+ 66,5 mL Mobile Phase A) was injected into the ÄKTA pure 25. Unwanted
fractions were eluted at first with 10 column volumes of 50% Mobile Phase B. Later the enzyme
sample was eluted in a linear gradient of 50% to 100% Mobile Phase B 10 column volumes. The flow
rate was 5 mL/min except during Loading (1 mL/min) and Elution (2.5 mL/min).
The Hydrophobic Interaction Chromatography was done twice and desired enzyme fractions of both
runs were pooled after SDS-PAGE.

2.2.2.2 SDS-PAGE
SDS-PAGE was performed on fractions from the HIC purification step to determine which fractions
should be further purified. 20 µL of each sample were mixed with 5 µL 5x Laemmli buffer. The mix
was incubated on a thermomixer (300 rpm) at 99 °C for 10 minutes. Then 10 µL of each sample were
loaded on Mini-Protean TGX Precast Gels. The SDS-PAGE was run for 45 minutes at 150 Volt using
Power Pac HV Power Supply. Once the run was over the gel was treated with Coomassie blue staining
solution. The gel was soaked in staining buffer for one hour on a shaker. Afterwards the used
solution was removed, destaining buffer was applied and left to soak on the shaker for another hour.
The destaining step was repeated four times. Pictures of the SDS-PAGE gel were taken after the run
using the stain free method und after the Coomassie treatment. The pictures were acquired using
the Biorad ChemiDoc.

2.2.2.3 Concentration
Desired enzyme fractions of both Hydrophobic Interaction Chromatography runs were pooled. This
pool was further concentrated from 90 mL to about 5 mL. This was achieved by centrifuging the pool
in Vivaspin 20 centrifugal concentrators with a molecular weight cut off (MWCO) of 3000 Dalton and
at 3700 rpm for 1h and 30 min.

2.2.2.4 Rebuffering
EndoB concentrate was rebuffered to 10 mM Tris buffer pH 7.5 for anion exchange chromatography.
First, PD-10 columns were filled with 10 mM Tris pH 7.5 buffer. The flow through consisting of 20%
ethanol storage solution was discarded. Once the columns were equilibrated with buffer, 2.5 mL
EndoB concentrate was added to each column. The sample solution was eluted from the column
using 3.5 mL 10 mM Tris buffer pH 7.5 and further purified with anion exchange chromatography.

2.2.2.5 Anion Exchange Chromatography (AEC)

A 5 mL HiTrap FF DEAE column from Cytiva was used for AEC purification. As binding buffer A 10 mM
Tris pH 7.5 was used. Elution buffer B consisted of 10 mM Tris pH 7.5 buffer with 1 M NaCl. The
column underwent several washing steps: First with Ethanol 20%, then with MQ-Water, and finally
with the buffers A and B. Afterwards, 30 ml EndoB sample was injected. The sample was eluted using
a linear gradient from 0% to 10% buffer B for 10 column volumes, followed by constant 10 % buffer B
phase for 5 column volumes. Desired enzyme fractions were identified with SDS-PAGE, pooled and
rebuffered to 50 mM citrate buffer (pH 4.) using PD-10 columns. Finally, the solution was
concentrated to a volume of 3 mL using Vivaspin 20 centrifugal concentrators

13
2.2.3 Protein concentration determination
To determine the specific activity of the enzymes, the protein content needed to be determined. This
was done by measuring the absorbance at 280 nm using the Nanodrop photometer as well as using
the Bradford Method.

2.2.3.2 Protein determination via Bradford Method

The protein content of various enzyme formulations was measured using the Bradford method. Pre-
diluted enzyme solutions (32 µL) were filled up with Bradford solution to 480 µL. 200 µL of each
sample where then transferred to a 96 well plate. The absorbance was measured at 595 nm. To
calculate protein content a calibration curve was calculated by measuring the absorbance of samples
containing BSA ranging from 0 to 0.4 mg/mL. Measurements were carried out in duplicates (n=2).

2.2.3.1 Protein determination via Nanodrop Photometry

The Implen NP80 Nanodrop was used to determine the photometric protein content of enzyme
dilutions. A volume of 2 µl of the enzyme sample was placed on the photometer sensor and the
protein concentration determined in mg/mL using the extinction coefficient of bovine serum albumin
(44, 289 M-1 cm-1).

2.2.4 Activity Assays

2.2.4.1 CellG5 Cellulase Activity Assay
The endoglucanase activity of the various enzyme formulations was measured using the CellG5
cellulase assay kit from Megazyme. For each enzyme formulation duplicate samples were prepared
(n=2). 100 µL substrate solution was incubated for 10 minutes at 45 °C with 100 µL enzyme solution.
The reaction was stopped with the addition of 3 mL stopping reagent (2% Tris solution pH 10). The
substrate solution contains β-glucosidase and blocked 4-nitrophenyl--D-cellopentoside.
Endoglucanase hydrolyses the 4-nitrophenyl--D-cellopentoside. The -glucosidase present in the
substrate solution degrades those hydrolytes and sets the 4-nitrophenol free. The absorbance at 400
nm was measured using a Hitachi U2900 photometer and the cellulase activity calculated in U/mL.

2.2.4.2 XylX6 Xylanase activity assay

Endo-Xylanase activity of EndoB was measured using the XylX6 xylanase assay kit from Megazyme.
The substrate solution contains -xylosidase and a derivatized xylopentaoside blocked with a ketone
group at the non-reducing end, which is hydrolyzed by xylanase. The -xylosidase present in the
substrate solution degrades those hydrolytes and sets the 4-nitrophenol free. 50 µL substrate
solution was incubated for 10 minutes at 45 °C with 50 µL enzyme solution (1:50 diluted). The
reaction was stopped with the addition of 1.5 mL stopping reagent (2% Tris solution pH 10).
Absorbance was measured at 400 nm using a Hitachi U2900 photometer. Xylanase activity was
calculated in U/mL from the absorbance at 400 nm.

2.2.4.3 Filter paper cellulase activity assay

The cellulase activity of the enzyme samples was calculated by incubation of the enzymes with
Whatman No. 1 filter paper and measuring the released glucose. Enzymes were diluted in 50 mM
citrate buffer at pH 4.8. First 0.75 cm by 7.5 cm filter paper strips were put in Pyrex glass tubes and
suspended in 400 µL citrate buffer. The reaction was started by addition of 100 µL enzyme dilution
at 45 °C. The reaction was stopped after 0 min (500 µL 1M NaOH were added before the enzyme
solution), 5, 10, 20, 40, and 60 min using 500 µL of 1M NaOH solution. For each time point duplicate
samples were prepared (n=2). Then 500 µL DNS solution was added to start the coloring reaction.
The tube’s lid was screwed tight, and the tube placed in a boiling water bath for 5 minutes.
14
Afterwards, 200 µL of each sample were transferred onto a 96 well plate for absorbance
determination. Measurements were taken in duplicates at 540 nm wavelength using a Tecan Infinite
M200 Pro photometer.

2.2.4.4 Cellulase activity assay on pulps with DNS

The cellulase activity of enzyme samples on different pulps was measured by incubating them with
pulps and then measuring the released glucose with a photometer. First 10 mg pulp samples were
placed in Pyrex glass tubes together with 200 µL of 50 mM Citrate buffer pH 4.8. The glass tubes
were heated to 45 °C. The reaction was then started by adding 50 µL of enzyme solution. The
reaction was stopped after 0 (blank), 10, 20, 40, and 60 minutes by addition of 250 µL 1M NaOH.
Then 250 µL DNS solution were added to the reaction mix to start the coloring reaction. The tube’s
lid was screwed tight, and it was placed in boiling water for 5 minutes. Finally, 200 µL of each sample
were transferred onto a 96 well plate for absorbance determination. Measurements were taken in
duplicates at 540 nm wavelength using a Tecan Infinite M200 Pro photometer.

2.2.4.5 -Glucosidase activity assay

-Glucosidase activity of the enzyme cocktails and purified EndoB endoglucanase was measured. For
every enzyme mixture, Pyrex glass tubes were prepared and filled with 200 µL of substrate solution
(2mM 4-Nitrophenyl β-D-glucopyranoside dissolved in 50 mM Citrate buffer pH 4.8). The substrate
was then equilibrated at 45 °C and 50 µL diluted enzyme solution was added to start the reaction.
Finally, the reactions were stopped after reacting for 0 min (Blank), 10, 20, 30, 40, 50 and 60 min at
45°C. This was done by adding 500 µL methanol to the reaction mixture. The only exception to this
was the EndoB purified endoglucanase. In that case the reaction was stopped at 0, 20, 40 and 60 min
to save enzyme solution. The colorimetric reaction was performed by adding 750 µL Na-PO4 buffer
(pH 7) to the Pyrex tubes. Finally, 200 µL of each sample were transferred onto a 96 well plate for
absorbance determination. Measurements were taken at 410 nm wavelength using a Tecan Infinite
M200 Pro photometer.

2.2.5 Fourier-transform infrared spectroscopy (FT-IR)

Samples of the short fiber pulp SF Kraft 1 (50 mg) were treated for four hours at 45 °C at 500 rpm on
a thermomixer with the original enzyme formulations. In addition, samples treated in the same way
but with desalted enzyme formulations. This was done with 1 Unit (according to the endoglucanase
specific CellG5 assay) of enzyme activity. The exception being Ctec3 which was done with 10 Units.
The reaction was stopped by incubation at 99°C for 5 min. FT-IR spectra were recorded using a Perkin
Elmer Spectrum 100 spectrometer at a resolution of 4 cm-1 ranging from 600 to 4000 cm-1. Untreated
pulp samples were measured as blank. All measurements were taken triplicates (n=3). The resulting
spectra were normalized at 1200 cm-1.

2.2.6 Scanning Electron Microscopy (SEM)

To measure the morphological changes of enzyme treatment on pulp samples, pictures were
acquired using the scanning electron microscope. A Hitachi TM3030 scanning electron microscope
was used. First samples of enzyme treated pulp were coated with 1 nm of platinum using a Leica EM
ACE 200 sputter coater. Then pictures were taken at 1000 times magnification. This was done at two
independent spots of each pulp sample. The resulting images were compared to equivalent pictures
from an untreated pulp sample.

2.2.7 Size Exclusion Chromatography (SEC)

To determine the changes in molecular weight by various enzyme formulations,
carboxymethylcellulose substrates were incubated with enzyme formulations. The resulting samples
15
were then examined using size exclusion chromatography. For each sample 25 µL enzyme solution
were added to 250 µL substrate (0.5 % CMC, DS=0.9, Molecular weight= 250000 g/mol in 50 mM
citrate buffer pH 4.8). In the case of the blanks 25 µL citrate buffer (50 mM, pH 4.8) were added
instead of enzyme solution. This reaction mix was then incubated at 45 °C for 2 hours. Afterwards the
samples were heated on a thermomixer at 99 °C for 15 minutes for inactivation. The samples were
then diluted 1: 2.5 with MQ water and filtered into a chromatography vial through a 0.45 µm nylon
filter. Size exclusion chromatography was performed using a 1260 Infinity II liquid chromatography
system from Agilent Technologies with a quaternary/binary pump and an autosampler (1260 series).
A MALLS HELIOS DAWN 2 detector from Wyatt Technology, as well as a diode array detector and a
refractive index detector were used for detection. The mobile Phase was 50 mM NaNO3 buffer with 3
mM NaN3. For separation, a PL aquagel-OH MIXED H (PL1549-5800, 4.6 × 250 mm2, 8 μm) column
was used together with a PL aquagel-OH MIXED Guard (PL1149-1840, 8 μm, 7.5 × 50 mm2) pre-
column. The resulting data was analyzed using the ASTRA 7 software. A volume of 100 µL of each
sample was injected and run for 90 minutes. The flow rate was 0.3 mL/min. Measurements were
performed in duplicates (n=2).

2.2.8 High Performance Liquid Chromatography (HPLC)

The collected supernatants of the incubation of the SF Kraft 1 pulp with enzyme formulations at a
dosage of 1 U (see 2.2.5) were analyzed with HPLC. The supernatant was diluted 1:5 with MQ-water.
Carrez-precipitation was commenced afterwards for removal of lipids and proteins. 20 µL C1 (2%
K4[Fe(CN)6] * 3 H2O) solution was added, the sample was vortexed and left to incubate for 1 minute.
Then 20 µL of C2 (2% ZnSO4 * 7 H2O) solution was added, the sample was again vortexed and left to
incubate for another 5 minutes. The now precipitated proteins were removed by centrifuging the
samples at 12500 rpm for 30 minutes. The now purified supernatant was filtered through a 0.45 µm
filter directly into HPLC vials. The HPLC system used was an Agilent 1260 Infinity LC system equipped
with an ION-300 column. The flow rate was 0.325 mL/min, and the separation time was 45 minutes.
Standards ranging from 0 to 0.96 mg/ml were run along with the samples and used for the
calculation of the concentration of the samples in mg/ml.

16
3 Results and discussion
3.1 Basic characterization of the enzyme formulations
The original enzyme formulations were characterized regarding their individual enzyme activities and
protein content. The same was done for their desalted forms. All enzyme formulations lost part of
their protein content during desalting.
EnzA had by far the lowest protein content, according to NanoDrop (before desalting 15.75 mg/mL,
after desalting: 2.805 mg/mL, 18% of its initial protein content). The commercial endoglucanase
FiberCare R showed the next highest protein content of 35.20 mg/mL before desalting and 28.5
mg/mL after (81% of original protein content). The highest protein content was measured with EnzC
with 298.95 mg/mL before and 147.6 mg/mL after desalting (49% of its original amount), which
suggests a high number or concentration of enzymes. A detailed overview over the measured protein
content using Nanodrop is highlighted in Figure 2.

Protein content of refining enzyme formulations

FiberCare R

EnzA
enzymes

EnzB

EnzC

0 50 100 150 200 250 300 350

protein content [mg/mL]

Desalted Salted

Figure 2 Protein content of the refining enzyme formulations according to Nanodrop photometry. Error bars indicate
standard deviation of the duplicates.

It is expected that the volumetric enzyme activity (U/mL) is lower in the desalted enzyme
formulations as some protein loss is occurring during desalting. However, because the salts were
removed, and it is unclear how those affected enzyme activity (as some salts may increase or
decrease enzyme activity). Therefore, the specific enzyme activity is of interest. To test these
assumptions the refining formulations, and their desalted forms, were tested for their endo-1,4-β-
glucanase activity, their cellulase activity, and their β-glucosidase activity.

Endoglucanase activity on CellG5

As expected, the desalted formulations had a lower volumetric endoglucanase activity than their
salted counterparts (see Fig. 3).

17
 Enzyme formulations (CellG5)

FiberCare R

EnzA

EnzB

EnzC

0 20 40 60 80 100 120 140 160 180

Endo-1,4-β-glucanase activity [Unit/mL]

Desalted Salted

Figure 3: Enzyme activities of enzyme formulations determined using CellG5. Error bars indicate standard deviation of the
duplicates.

However, except for FiberCare R, the specific endoglucanase activity of all enzyme formulations
increased after desalting (Figure 4). Only FiberCare R saw a decline in specific enzyme activity after
desalting. The most significant increase in specific endoglucanase activity was measured with EnzA. It
increased 2.55-fold, from 0.236 Unit/mg to 0.603 U/mg. The next steepest increase was that of EnzC
with a 1.85-fold higher activity after desalting (from 0.5485 U/mg to 1.01 U/mg). The lowest increase
of specific endoglucanase activity was that of EnzB (1.16-fold). FiberCare R’s specific endoglucanase
activity even decreased, from 1.871 U/mg to 1.098 U/mg, which could indicate that salts were added
to this formulation that enhance endoglucanase activity and are now removed.

Enzyme formulations (CellG5)

FiberCare R

EnzA

EnzB

EnzC

0 0.5 1 1.5 2
Specific Endo-1,4-β-glucanase Activity [Unit/mg]

Desalted Salted

Figure 4: Specific enzyme activities of enzyme formulations determined using CellG5. Error bars indicate standard deviation
of the duplicates.

Dissolved salts change the enzyme activity. Salts can influence the flexibility of the catalytic center
(simultaneously stabilizing the enzyme) or destabilize the enzymes through interaction with their
amino acid chain. So, there is a concentration of dissolved salts that is ideal for the enzymes, also the
composition of dissolved salts is of importance. EnzC, EnzB and EnzA may be closer to their optimum

18
after desalting than before. In contrast FiberCare R was closer to its ideal state before desalting.
Furthermore, enzymes can agglomerate at high concentrations and impair enzyme activity. All
refining enzyme formulations have lost protein content during desalting, which could be related to
this effect.

Filter paper cellulase assay

The action of cellulases was investigated on filter paper. For EnzC the volumetric cellulase activity
increased after desalting despite the protein loss (Figure 5). It increased from 10.65 U/mL to 14.84
U/mL, a 1.39-fold increase. The cellulase activity per mL of EnzC increased while its endoglucanase
activity on CellG5 decreased after desalting. This can be explained by the function of the filter paper
assay as it measures released reducing sugars such as glucose. Endoglucanase cut the cellulose
chains, thereby releasing cello-oligosaccharides. Other enzymes, like β-glucosidase, can then degrade
the released cello-oligosaccharides into glucose units, thus increasing the signal. Therefore, a higher
β-glucosidase activity of desalted EnzC could explain the discrepancy. However, the specific activity
of EnzC In U/mg was also higher on CellG5, suggesting an increase in endoglucanase activity after
desalting (Figure 4), but the increase was even higher on filter paper, which supports the
contribution of β-glucosidase. EnzB and FiberCare R decreased in cellulase activity after desalting on
filter paper (12.75 U/mL to 11.1 Unit/mL and from 3.98 U/mL to 3.52 U/mL, respectively), while the
activity of EnzA remained relatively stable (declining from 0.3010 U/mL to 0.2977 U/mL).

Refining enzyme formulations

(Filter paper cellulase activity assay)

FiberCareR
Enzymes

EnzA

EnzB

EnzC

0 2 4 6 8 10 12 14 16 18
Cellulase activity [Unit/mL]

Desalted Salted

Figure 5: Cellulase activities of refining enzyme formulations according to filter paper assay. Error bars indicate standard
deviation of the duplicates.

The specific cellulase activity increased with desalting for every enzyme formulation (Figure 6). In the
case of FiberCare R the increase was only marginal. Before desalting, specific enzyme activity was
0.113 U/mg, after desalting 0.123 U/mg. EnzA saw the biggest increase in specific enzyme activity.
Before desalting its activity was 0.019 U/mg and afterwards 0.105 U/mg (a 5.53-fold increase). The
increase in specific activity for EnzB and EnzC was more moderate. The specific activity of EnzC
increased from 0.036 U/mg to 0.101 U/mg after desalting (a 2.81-fold increase), while the specific
activity of EnzB increased from 0.068 U/mg before desalting to 0.100 U/mg after desalting.

19
 Enzyme formulations
(Filter paper cellulase activity assay)

Fiber Care R
Enzymes

EnzA

EnzB

EnzC

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Specific cellulase activity [Unit/mg]

Desalted Salted

Figure 6: Specific cellulase activities of enzyme formulations according to filter paper assay. Error bars indicate standard
deviation of the duplicates.

β-glucosidase activity

β-glucosidase activity of desalted EnzC is much higher than that of salted EnzC (Figure 7). In fact, it
increased 4.06-fold from 4.367 U/mL to 17.74 U/mL. EnzC also had the overall highest β-glucosidase
activity, EnzA the lowest. The β-glucosidase activity of EnzB decreased after desalting from 3.169
U/mL to 2.24 U/mL (a decrease to 71% of the original activity.), which could explain its lower
performance in the filter paper assay. Meanwhile the activities of EnzA and FiberCare R remained the
same with only slight changes. FiberCare R had the lowest overall β-glucosidase activity, which is
unsurprising considering, FiberCare R is an endoglucanase formulation and does not include other
enzymes than endoglucanases.

β-glucosidase activity of enzyme formulations

FiberCare R

EnzA
enzyme

EnzB

EnzC

0 5 10 15 20
β-glucosidase activity [Units/mL]

Desalted Salted

Figure 7: β-glucosidase activities of refining enzyme formulations. Error bars indicate standard deviation of the duplicates.

The specific β-glucosidase activity of EnzA and EnzC increased after desalting (Figure 8). The highest
increase was measured with EnzC (0.014 U/mg to 0.120 U/mg), an 8.57-fold increase. The specific
20
activity of EnzA was 0.047 U/mg before desalting and 0.267 U/mg after (5.68-fold increase). The
increase of activity after desalting of EnzB was only moderate a 1.18-fold increase). The specific β-
glucosidase activity of FiberCare R was as expected neglectable.

β-glucosidase activity of enzyme formulations

Fiber Care R

EnzA
enzymes

EnzB

EnzC

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

specific β-glucosidase activity [Units/mg]

Desalted Salted

Figure 8: specific β-glucosidase activities of refining enzyme formulations. Error bars indicate standard deviation of the
duplicates.

3.2 Basic characterization of purified EndoB endoglucanase

The purified EndoB endoglucanase was characterized regarding its individual enzyme activities and
protein content. The final protein content of EndoB was adjusted to 7 mg/mL (7.152 mg/mL). For
comparison, the unpurified EnzB enzyme mix had a protein content of 25.108 mg/mL (3 times higher
than purified EndoB). The protein content of EndoB was also measured using the Bradford method,
giving a value of 1.13155 mg/mL. The difference is caused by Nanodrop and Bradford being based on
two different methods.

Endo B was tested for its endoglucanase activity, its cellulase activity, its endoxylanase activity, and
its β-glucosidase activity. Its endoglucanase activity was 0.9325 U/mL according to CellG5-assay.
Cellulase activity was determined to be 0.260 U/mL according to filter paper cellulase activity assay,
while its Endo-xylanase activity was 10.34 Units/mL and the β-glucosidase activity 0.0446 U/mL.

3.3 Enzymatic treatment of different pulps using EndoB

Cellulase activity of EndoB was tested on different pulps. The tested pulps were Short Fiber kraft
pulp, Short Fiber kraft Pulp 2, Long Fiber kraft pulp, and Long Fiber sulfite pulp (see Fig. 9).

21
 Cellulase activity of EndoB
on various pulps

SF Kraft 1

SF Kraft 2
Pulps

LF Kraft

LF Sulfite

0 0.05 0.1 0.15 0.2 0.25

Cellulase activity [Unit/mL]

Figure 9: Cellulase activity for EndoB, concentrated to 7 mg/mL, on various pulps. Error bars indicate standard deviation of
the duplicates.

On average, EndoB exhibited a higher cellulase activity on short fiber pulp than long fiber pulp.
Within those categories it hydrolyses short fiber pulp 2 (0.199 U/mL) better than pulp 1 (0.1413
U/mL). Among the longs fiber pulps, sulfite pulp was better hydrolyzed (0.1477 U/mL) than sulfate
pulp (0.0830 Units/mL).

3.4 Enzymatic treatment of substrates with commercial enzyme formulations

3.4.1 HPLC
For Evaluation of the released products of the enzyme formulations, the supernatant from enzyme
treated SF sulfate pulp samples was analyzed with HPLC after incubation with 1 U of enzyme activity
(CellG5).

Every enzyme cocktail, both salted and desalted, released more Xylose than any other component
(highest by EnzB: before desalting: 0.130 mg/mL, after desalting: 0.133 mg/mL). FiberCare R had
quite different HPLC results from the other enzyme formulations (Figure 10). FiberCare R didn’t
release any xylose at all as expected for an endoglucanase formulation. All three refining enzyme
formulations released comparable amounts of glucose. The glucose release of EnzC decreased with
desalting (0.065 mg/mL to 0.055 mg/mL), while that of EnzB remained constant (0.0490 mg/mL to
0.0485 mg/mL) and that of EnzA increased (0.051 mg/mL to 0.065 mg/mL). None of the three
refining enzyme formulations produced high quantities of Cellobiose, Cellotriose or Cellotetraose.
Cellobiose was released the most by FiberCare R (0.024 mg/mL before desalting and 0.032 mg/mL
after desalting) and EnzB (0.012 mg/mL to 0.007 mg/mL). Cellotriose was also released in high
amounts by FiberCare R (0.0082 mg/mL before desalting and 0.0090 mg/mL after desalting) and EnzB
(before desalting: 0.0047 mg/mL, after desalting: 0.0050 mg/mL). The highest amount of
Cellotetraose was released by EnzA (salted: 0.0092 mg/mL; desalted: 0.0070 mg/mL), the lowest by
EnzB.

22
 HPLC
supernatant of enzyme treated SF Kraft 1 pulp treated
with 1 Unit enzyme
0.16
Released sugars [mg/mL]

0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
EnzC EnzC EnzB EnzB Fiber Care Fiber Care Enz A EnzA
desalted desalted R R desalted desalted
Refining enzyme formulations

Glucose [mg/ml] Xylose [mg/ml] Cellobiose [mg/ml]

Cellotriose [mg/ml] Cellotetraose [mg/ml]

Figure 10: Release of different sugers through treatment of SF Kraft 1 pulp with 1 Unit enzyme formulations. Error bars
indicate standard deviation of the duplicates.

3.4.2 Scanning Electron Microscopy

To analyze the effects of enzyme treatment on morphology of pulps, Short Fiber Kraft 1 pulp samples
were treated with 1 Unit (CellG5) of enzyme formulations. Afterwards pictures were acquired using a
Scanning Electron Microscope at 1000 times enlargement. Those pictures were compared to an
untreated blank sample (Figure 11). In addition, the effect of desalting was analyzed (Figure 12):

Comparison of the effect of enzyme formulations

Figure 11: Samples of Short Fiber Kraft 1 pulp treated with 1 Unit (CellG5) salted enzyme formulations. The pictures were
acquired with a Scanning Electron Microscope at 1000 times enlargement.

23
EnzA treated fibers appear damaged towards the end as if ripped apart and parts seem to be peeling
of along the fiber. Similar fiber diameter was observed when compared to the untreated sample.
There are very few longitudinal and transverse notches. The fibers treated by EnzB are thinner
compared to the blank sample and little holes within the fiber were observed. The treated sample
shows more fiber damage caused by enzyme treatment. There are also more transverse notches and
fibers are getting thinner towards the end. The EnzC treated pulp sample has fiber tips that get
thinner towards the end compared to the untreated sample and the fibers appear thinner in general.
FiberCare R resulted in fibers that are much thinner compared to the blank sample. Fiber damage is
present but rare and there are noticeable longitudinal notches and transversal notches along the
fiber.

Comparison of the effect of salted vs. desalted enzyme formulations

Figure 12: Samples of Short Fiber Kraft 1 pulp treated with 1 Unit (CellG5) salted enzyme formulations. The pictures were
taken with a Scanning Electron Microscope at 1000 times enlargement.

EnzC desalted treated fibers have a similar thickness as the ones treated with regular EnzC. In
addition, these fibers have more and deeper longitudinal notches. The fibers appear to be getting
thinner towards the end and some fiber damage is visible. The EnzB desalted treated sample has less
fiber damage than the one treated with salted EnzB. Fibers are still growing thinner towards the tip
and there are also less notches. The EnzA desalted treated sample is similar to salted EnzA but has
more fiber damage, which is however, less extreme at the fiber ends. The longitudinal notches on
FiberCare R desalted treated fibers are less pronounced than on the regular FiberCare R sample. The
fibers are growing thinner towards the tip and there is hardly any fiber damage visible.

24
Figure 13: A sample of Short Fiber Kraft 1 pulp treated with 10 Units of CTec3. The pictures were taken with a Scanning
Electron Microscope at 1000 times enlargement.

For comparison, another Short Fiber Kraft 1 sample was treated with 10 Units of the formulation
CTec3, a hydrolytic enzyme cocktail capable of degrading cellulose fibers efficiently into glucose
(Figure 13). The fibers are partially dissolved and remaining fibers are thinner than the blank.
Prominent holes and longitudinal notches are apparent.

3.4.3 Size Exclusion Chromatography

Carboxymethylcellulose with an degree of substitution and an average molecular weight of 250.000
g/mol was treated with enzyme formulations and pure endoglucanases to evaluate how treatment
with different enzymes would modify the weight average molecular weight ( Mw ) and dispersity

( Mw / Mn ) after analysis with size exclusion chromatography.

The blank without enzyme treatment was used for comparison of the enzyme effect and it exhibited
a molecular weight of 1.74*106 g/mol, while its dispersity index was 3.23 (Table 1). Treatment with
EnzA and EnzC led to the overall lowest weight average molecular weight of only 1.07*105 g/mol,
suggesting the generation of the overall smallest fragments.

The dispersity of the EnzA fragments was slightly higher (2.22) than that of the EnzC fragments
(2.12). Therefore, the distribution of the fragment sizes was a lot narrower for EnzC. However, EnzB
exhibited the lowest overall dispersity index of 2.08, while the determined weight average molecular
weight was with 1.24*105 g/mol higher than EnzA and EnzC, suggesting the degradation of small
fragments. The purified endoglucanases all exhibited higher molecular weights, when compared to
the enzyme formulations containing different enzymes. The EndoB endoglucanase sample contained
the smallest fragments with 1.80*105 g/mol molecular weight and a dispersity index of 2.28,
followed by EndoA with a molecular weight of 1.87*105 g/mol and a dispersity index of 2.18.
Treatment with EndoC produced the largest fragments., their weight average molecular weight was
2.15*105 g/mol. The purified endoglucanases do not contain β-glucosidases, therefore higher
molecular weight fragments cannot be degraded into smaller fragments without these enzymes.

CTec3 produced slightly larger fragments with a weight average molecular weight of 1.61*105 g/mol,
despite it is a hydrolytic enzyme formulation. However, this effect could be related to the dosage
according to the endoglucanase specific CellG5 assay.

25
Table 1: Weight average molecular weight Mw and dispersity index of enzyme treated samples.

Description (treatment Mw Mw
duration) [g/mol] / Mn
EnzA (2h) 1.07×105 2.22
EnzB (2h) 1.24×105 2.08
5
EnzC (2h) 1.07×10 2.12
FiberCare R (2h) 1.28×105 2.25
EnzC Puri (2h) 2.15×105 2.33
EnzB puri (2h) 1.80×105 2.28
5
EnzA puri (2h) 1.87×10 2.18
Ctec3 (2h) 1.61×105 2.28
Blank (with buffer)
1.74×106 3.23
(0min)

3.4.4 FT-IR
Change of cellulose properties such of crystalline and amorphous regions of enzyme treated SF Kraft
1 pulp samples were analyzed with a Fourier Transformation-Infrared spectrometer. Moreover, the
effect of salted and desalted enzyme formulations was evaluated (see Figures 14 and 15) and
characteristic parameters such as the Total crystallinity index (TCI), the Hydrogen bond intensity (HBI)
and the absorbance at the amorphous region were calculated. The obtained spectra, absorbance
values and calculated values are highlighted in Figures 18, 19 and 20 (see appendix) as well as Tables
2, 3 and 4.

26
 FT-IR spectra of enzyme treated pulp
12
samples (salted)
10

8
Absorbance

0
650 1150 1650 2150 2650 3150 3650
Wave number [cm-1]

Blank EnzC FiberCare R EnzA EnzB

Figure 14: FT-IR spectra of enzyme treated SF Kraft 1 pulp samples. The samples were treated with 1 Unit (CellG5) enzyme
activity for 4 hours at 45 °C.

FT-IR spectra of enzyme treated pulp samples

(desalted)
12

10

8
Absorbance

0
650 1150 1650 2150 2650 3150 3650
Wave number [cm-1]

Blank EnzC desalted FiberCare R desalted EnzB desalted EnzA desalted

Figure 15: FT-IR spectra of enzyme treated SF Kraft 1 pulp samples. The samples were treated with 1 Unit (CellG5) enzyme
activity for 4 hours at 45 °C.

27
Amorphous region

Endoglucanases can only degrade amorphous regions. Therefore, lower absorbance at 897 cm-1,
which corresponds to the amorphous region, indicates degradation by endoglucanases. This could
translate to a more efficient refining. However, dosage during refining is usually at low levels. With
the dosage of 1 U, the effect of enzyme overdosing was evaluated. EnzA resulted in the lowest
absorbance at 897 cm-1 with a value of 2.52 (Table 2). Desalted EnzA had a very similar result with an
absorbance of 2.93. Of the remaining three formulations EnzC performed best (absorbance: 3.1364),
followed by FiberCare and EnzB. Interestingly, EnzB was the only enzyme formulation that performed
better after desalting. Treatment with desalted EnzB led to an absorbance at 897 cm-1 of only 2.67. In
contrast, FiberCare R led to comparable results and EnzC became less efficient with desalting.
Table 2: Absorbance at amorphous region 897 cm-1

Absorbance at amorphous region 897 cm-1

SF Kraft 1 Blank
4.72
EnzC EnzC desalted
3.14 4.46
FiberCare R
FiberCare R desalted
3.42 3.27
EnzA EnzA desalted
2.52 2.93
EnzB EnzB desalted
3.46 2.67

Total crystallinity index

The total crystallinity index measures changes in crystallinity of cellulose fibers. Since endoglucanases
usually only can degrade amorphous cellulose, an increase in crystallinity is expected. However, this
can change if the enzyme cocktail used to refine the cellulose fibers also contains β-glucosidases.
Those can degrade the layers of cellulose from pulp, thus reverting absorbance values to the
“starting value”.

All enzyme treated samples resulted in a higher total crystallinity index than the blank (Table 3).
The total crystallinity index increased with desalting for EnzA and EnzB while it decreased for EnzC
and FiberCare R, which could indicate disruption of the crystal structure. The highest total
crystallinity index was measured with EnzC (salted) and EnzB (desalted). Treatment with salted
FiberCare R resulted in a total crystallinity index of 2.25, however desalting led to the lowest
measured TCI of all enzyme formulations: 2.09, thereby promoting salts could have been removed.
EnzA had the lowest effect on the TCI in its original form (2.09), however desalting led to a similar
value (2.32) as EnzC.

28
Table 3: Total crystallinity index (TCI)

Total crystallinity index (TCI)

(Nelson and O'Connor) = ratio of 1372 and 2900
cm-1
SF Kraft 1 Blank
1.69
EnzC EnzC desalted
2.49 2.33
FiberCare R FiberCare R desalted
2.25 2.09
EnzA EnzA desalted
2.09 2.32
EnzB EnzB desalted
2.38 2.59

Hydrogen bond intensity

The hydrogen bond intensity (HBI) correlates with the crystallinity of the pulp sample and with bound
water. Hydrogen bonds connect the cellulose fiber together and a higher amount of hydrogen bonds
implies higher crystallinity. All enzyme treated samples had a lower hydrogen bond intensity than the
blank (Table 4).

The highest value was indeed caused by EnzA (0.69) and FiberCare R (0.67) in their original form. The
EnzC and EnzB treated samples exhibited the lowest HBI while having the highest TCI. However, the
Hydrogen Bond Intensity of cellulose is correlated both to the amount of bound water and to the
degree of crystallinity (CHAMBRE & DOCHIA, 2021). Therefore, it is expected that the samples with
high TCI would also have high HBI, however instead the HBI decreased for all enzyme formulations.
One possible explanation for this could be due to the way the samples were treated. The
endoglucanases cut at the amorphous parts of the fiber, which induces fiber damage, weakens the
fiber, and can disrupt the hydrogen bond network within the cellulose fiber, resulting in a lower HBI,
while the crystalline parts of the sample fiber remain unharmed leading to a higher TCI.
Table 4: Hydrogen Bond Intensity (HBI)

Hydrogen bond intensity = ratio of 3400 and 1320

cm-1
SF Kraft 1 Blank
0.77
EnzC EnzC desalted
0.54 0.61
FiberCare R FiberCare R desalted
0.67 0.71
EnzA EnzA desalted
0.69 0.61
EnzB EnzB desalted
0.60 0.54

29
3.5 Composition of enzyme formulations
To visualize the enzyme content of the enzyme formulations and pure endoglucanases an SDS-PAGE
was performed (Figure 16). For comparison, another gel was run with purified versions of EnzA, EnzB
and EnzC (EndoA, EndoB and EndoC).

Figure 16: SDS-Page of enzyme formulations and purified EndoB.

EnzA/EndoA
EnzA has its main band between 25 and 15 kDa, while also having a sideband at 35kDa (Figure 16).
The main band is most likely the endoglucanase enzyme itself. This is confirmed by the analysis of the
purified endoglucanase EndoA, which lacks the sideband at 35 kDa, while the main band is much
bigger and broader (Figure 16). Moreover, there is a very faint sideband between 15 kDa and 10 kDa.
EnzB/EndoB
The main band of EnzB is between 40 kDa and 55 kDa (Figure 16). Moreover, there is a faint sideband
between 35 kDa and 25 kDa. Above the main band in the area between 55 and 70 kDa also several
smeared protein bands are visible. The same is true for the area beneath the sideband. Comparison
with the purified EndoB enzyme, reveals that the previously characterized large main band between
55 kDa and 40 kDa is indeed the endoglucanase. However, also three faint side bands are visible at
100 kDa, 70 kDa and between 35 and 25 kDa, which suggest some residual enzyme fractions could
not be entirely removed through purification.

EnzC/EndoC
A main band can be seen at 35 kDa. Additional faint bands are visible along its separation path,
suggesting additional enzymes. The EndoC purification separated much cleaner, with one big main
band at 35 kDa and two faint sideband (70 and 55 kDa), suggesting the 35 kDa band is the
endoglucanase.

FiberCare R
Only a single band is visible at 40 kDa, which was expected, because FiberCare R contains only
endoglucanase (Figure 16).
30
CTec3
CTec3 separates nicely into many different bands (Figure 16). There are two clearly defined bands at
130 kDa and 100 kDa. The biggest band is between 70 and 55 kDa. In summary, 8 bands could be
identified. The results highlight that there is a high number of different enzymes in the hydrolytic
enzyme formulation intended for complete degradation of cellulose.

31
4 Conclusion
In this study, enzyme formulations (EnzA, EnzB, EnzC, FiberCare R) were characterized and the
endoglucanase EndoB was purified. The tests were carried out with original and desalted
formulations.

In the CellG5 endoglucanase assay, all desalted enzyme formulations had a lower volumetric activity
(U/mL) than the original ones, which was expected because of protein losses during desalting.
However, all enzyme formulations showed higher specific activities (U/mg), expect FiberCare R,
however FiberCare R was still the enzyme formulation with the highest activity on CellG5. Similar
results were obtained in the filter paper assay with a lower volumetric activity for the desalted
formulations except for EnzC. However, specific cellulose activity on filter paper was much higher for
all enzyme formulations. This could be related to higher β-glucosidase activities after desalting, which
were higher for EnzC and EnzA.
Characterization of purified EndoB endoglucanase
Endoglucanase EndoB was purified for further study. The endoglucanase activity and the activity on
filter paper was lower than the EnzB formulation, but this is related to the lower protein content. In
addition, a rather high xylanase activity was determined, which suggests a side-activity of this
endoglucanase. The activity on four pulps was tested and showed highest results on SF Kraft pulp 2
and lowest on LF kraft pulp, which suggests a varying affinity of the enzyme.
Effect of enzymes on pulp:

Sugar release from SF kraft 1 pulp was analyzed by HPLC. EnzA, EnzB and EnzC (desalted and non-
desalted) released primarily xylose and glucose, while significantly less of the other sugars were
released. Desalted EnzA released more sugar than the original variant. For EnzB and EnzC, there was
only a slight difference between desalted and non-desalted variants. The SEM analysis of enzyme
treated SF Kraft 1 showed thinner fibers by all formulations, an increased number of grooves was
seen in the cellulose fibers and cut-off ends were visible for EnzA treated fibers. The molecular
weight and size distribution of enzyme treated carboxymethylcellulose was analyzed by size
exclusion chromatography. The EnzB treated sample had the lowest dispersity, possibly because
fragments were degraded by the high β-glucosidase activity. EndoB without β-glucosidases had the
highest dispersity, and thus the widest distribution. The lowest molecular weights were obtained
with EnzA and EnzC formulations, while EndoC had the highest, which might be also related to
removed β-glucosidase activity of this purified endoglucanase. Changes to pulp properties were
measured using FT-IR. The absorbance at the amorphous region was lowest with non-desalted EnzA
and EnzC. However, desalting resulted in highest absorbance values for EnzC and lowest for EnzB,
highlighting the importance of the appropriate salt conditions. This correlates also with measured
TCI, which was highest with desalted EnzB. The HBI was most affected by EnzC and desalted EnzB,
which suggests a distribution of the hydrogen bonds by these enzymes.

In conclusion, the salt composition of enzyme formulations is a major factor for enzyme activity and
optimizations should be considered for most efficient pulp refining.

32
5 Appendix

Changes in molecular weight determined with SEC:

SEC
mass average molecular weight
2.5
mass average molecualr weight [g/mol]

1.5

0.5

0
EnzA EnzC EnzB FiberCare Ctec3 EnzB puri EnzA puri EnzC Puri
R
enzymes

Figure 17: Mass average molecular weight of CMC-substrate fragments after enzyme treatment.

Diagrams for visualization of FT-IR results:

Total crystallinity index (TCI)

3
TCI = ratio of absrobance at 1372 and

2.5
2
1.5
1
2900

0.5
0
EnzA EnzB EnzC FiberCare R Blank
Enzymes

Salted Desalted

Figure 18: Total crystallinity index of pulp samples after treatment with enzyme cocktails.

33
 Absorbance at amorphous region 897 cm-1
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
EnzA EnzB EnzC FiberCare R Blank

Salted Desalted

Figure 19: Absorbance at amorphous region of pulp samples after treatment with enzyme cocktails.

Hydrogen bond intensity

0.9
Hydrogen bond intensity = ratio of 3400

0.8
0.7
0.6
0.5
0.4
and 1320

0.3
0.2
0.1
0
EnzA EnzB EnzC FiberCare R Blank
Enzymes

Salted Desalted

Figure 20: Hydrogen bond intensity of pulp samples after treatment with enzyme cocktails.

34
Acknowledgements
First of all, I would like to thank Professor Gübitz for the opportunity to work on this project.
Secondly, I would like to thank my supervisor Martin for his good advice, tutelage and overall being a
great teacher. One can only wish every student to have such a supervisor.
Last but not least, I would like to thank my friends and family for their support during the writing of
this thesis.

35
6 References

Bajpai, P. (2018). Refining and Pulp Characterization. Biermann’s Handbook of Pulp and Paper, 1–34.
https://doi.org/10.1016/B978-0-12-814238-7.00001-5

Biermann, C. J. (1996). Chapter 3 - Pulping Fundamentals. Handbook of Pulping and Papermaking

(Second Edition), 55–100.
http://www.sciencedirect.com/science/article/pii/B9780120973620500078

CHAMBRE, D. R., & DOCHIA, M. (2021). FT-IR CHARACTERIZATION OF CELLULOSE CRYSTALLINITY

FROM RAW BAST FIBERS. Scientific and Technical Bulletin, Series: Chemistry, Food Science and
Engineering, 18, 10–17. https://uav.ro/jour/index.php/stb-cfse/article/view/1731

Haske-Cornelius, O., Hartmann, A., Brunner, F., Pellis, A., Bauer, W., Nyanhongo, G. S., & Guebitz, G.
M. (2020). Effects of enzymes on the refining of different pulps. Journal of Biotechnology, 320,
1–10. https://doi.org/10.1016/J.JBIOTEC.2020.06.006

KEGG ENZYME: 3.2.1.21. (n.d.). Retrieved June 23, 2022, from https://www.genome.jp/dbget-
bin/www_bget?ec:3.2.1.21

Kumar, A., Tazeb, A., & Ram, C. (2021). Enzyme-assisted pulp refining: An energy saving approach.
Physical Sciences Reviews, 6(2). https://doi.org/10.1515/PSR-2019-0046

Ochre Media Private Limited. (2018). Pulp and Paper Manufacturing Process in the Paper Industry. In
Pulp and Paper - Technology.com. Ochre Media Private Limited. https://www.pulpandpaper-
technology.com/articles/pulp-and-paper-manufacturing-process-in-the-paper-industry

Song, Z. (2018). Comparison Of Softwood Pulp And Hardwood Pulp | Paper Pulp Mill.
http://www.paperpulpingmachine.com/softwood-pulp-and-hardwood-pulp-comparison/

List of Figures
Figure 1: Overview of Pulp and Paper manufacturing. (Pulp and Paper Manufacturing Process | Steps in
Papermaking Procedure, n.d.) 6
Figure 2 Protein content of the refining enzyme formulations according to Nanodrop photometry. Error bars
indicate standard deviation of the duplicates. 17
Figure 3: Enzyme activities of enzyme formulations determined using CellG5. Error bars indicate standard
deviation of the duplicates. 18
Figure 4: Specific enzyme activities of enzyme formulations determined using CellG5. Error bars indicate
standard deviation of the duplicates. 18
Figure 5: Cellulase activities of refining enzyme formulations according to filter paper assay. Error bars
indicate standard deviation of the duplicates. 19
Figure 6: Specific cellulase activities of enzyme formulations according to filter paper assay. Error bars
indicate standard deviation of the duplicates. 20
Figure 7: β-glucosidase activities of refining enzyme formulations. Error bars indicate standard deviation of
the duplicates. 20
Figure 8: specific β-glucosidase activities of refining enzyme formulations. Error bars indicate standard
deviation of the duplicates. 21
Figure 9: Cellulase activity for EndoB, concentrated to 7 mg/mL, on various pulps. Error bars indicate
standard deviation of the duplicates. 22
Figure 10: Release of different sugers through treatment of SF Kraft 1 pulp with 1 Unit enzyme formulations.
Error bars indicate standard deviation of the duplicates. 23
36
Figure 11: Samples of Short Fiber Kraft 1 pulp treated with 1 Unit (CellG5) salted enzyme formulations. The
pictures were acquired with a Scanning Electron Microscope at 1000 times enlargement. 23
Figure 12: Samples of Short Fiber Kraft 1 pulp treated with 1 Unit (CellG5) salted enzyme formulations. The
pictures were taken with a Scanning Electron Microscope at 1000 times enlargement. 24
Figure 13: A sample of Short Fiber Kraft 1 pulp treated with 10 Units of CTec3. The pictures were taken with a
Scanning Electron Microscope at 1000 times enlargement. 25
Figure 14: FT-IR spectra of enzyme treated SF Kraft 1 pulp samples. The samples were treated with 1 Unit
(CellG5) enzyme activity for 4 hours at 45 °C. 27
Figure 15: FT-IR spectra of enzyme treated SF Kraft 1 pulp samples. The samples were treated with 1 Unit
(CellG5) enzyme activity for 4 hours at 45 °C. 27
Figure 16: SDS-Page of enzyme formulations and purified EndoB. 30
Figure 17: Mass average molecular weight of CMC-substrate fragments after enzyme treatment. 33
Figure 18: Total crystallinity index of pulp samples after treatment with enzyme cocktails. 33
Figure 19: Absorbance at amorphous region of pulp samples after treatment with enzyme cocktails. 34
Figure 20: Hydrogen bond intensity of pulp samples after treatment with enzyme cocktails. 34

List of Tables
Table 1: Weight average molecular weight Mw and dispersity index of enzyme treated samples. 26
Table 2: Absorbance at amorphous region 897 cm -1 28
Table 3: Total crystallinity index (TCI) 29
Table 4: Hydrogen Bond Intensity (HBI) 29

37